Immunogenic meningococcal LPS and other membrane vesicles and vaccine therefrom

ABSTRACT

The invention is directed to an immunity providing B cell activating molecule derived from a meningococcal lipopolysaccharide (LPS) having at least one epitope, said molecule comprising at least the communal part of the oligosaccharide part (core region) of lipopolysaccharides specific for at least two meningococcal immunotypes, preferably immunotypes L2 and L3 and wherein in galactose is absent in the B cell activating part, as well as derivatives of the molecules with immuno reaction inducing capacity. The invention is also directed at an outer membrane vesicle provided with a group of polypeptides having at least the immunoactivity of outer membrane proteins (OMP&#39;s) bound to a membrane, a polypeptide from the group of said outer membrane vesicles being a membrane anchored OMP or OMP fragment with a mutation in one of the surface loops, preferably in a 2, 3, 5, 6, 7 or 8-loop of a class I OMP. Furthermore, the invention is directed at a vaccine comprising such an outer membrane vesicle and/or lipopolysaccharide, as well as methods for preparing a lipopolysaccharide and an outer membrane vesicle as described above.

The subject invention is directed at an immunity providing B cellactivating molecule derived from a meningococcal lipopolysaccharide(LPS), said molecule comprising at least one epitope, said moleculecomprising at least the common portion of the oligosaccharide portion(core region) of lipopolysaccharides which are specific for at least twomeningococcal immunotypes. The invention is also directed at methods forthe preparation thereof, both synthetically and throughrecombinant-DNA-techniques. Furthermore, the subject invention isdirected at an outer membrane vesicle provided with a group ofpolypeptides which possess at least the immuo activity of outer membraneproteins (OMP's) bound to a membrane. The invention is also directed ata vaccine comprising such a molecule and/or such an outer membranevesicle. A method for preparing such an outer membrane vesicle alsofalls within the scope of protection of the subject invention.

It is known that vaccines of purified capsular polysaccharides (CPS) caninduce protective immunity. This immunity depends on the age of thevaccinated person and is only of a short duration. The intrinsicdisadvantages of capsular polysaccharides as vaccines are circumventedby the classic approach of coupling capsular polysaccharides oroligosaccharides to be derived therefrom with proteins (Goebel, W. F.,and O. T. Avery 1929 J. Exp. Med. 50:533-550 and Cruse, J. M. and R. E.Lewis (Ed.) 1989 Contrib. Microbiol. Immunol. Basel. Krager, 10:1-196).The coupling of polysaccharides to proteins results in changing thecharacter of this type of antigen from thymus independent to thymusdependent. Such poly or oligosaccharide protein conjugates are ingeneral very immunogenic in young children and can induce memory.

A number of examples of known saccharide peptide conjugates follow:

Conjugates of capsular polysaccharides (CPS) of H. influenza b withtetanus toxoid (TT) are known from the Dutch Patent Application 8602325.

Conjugates of capsular polysaccharides of meningococcal group A and Cwith tetanus toxoid were prepared, said conjugates appear to be veryimmunogenic in mice and rabbits ( Beuvery, E. C., A. Kaaden, V. Kanhaiand A. B. Leussink, 1983. Physicochemical and immunologicalcharacterization of meningococcal group A and C polysaccharide-tetanustoxoid conjugates prepared by two methods. Vaccine 1:31-36!, Beuvery, E.C., F. Miedema, R. van Delft and K. Haverkamp, 1983. Preparation andimmunochemical characterization of meningococcal group C polysaccharidetetanus toxoid conjugates as a new generation of vaccines. Infect.Immun. 40:39-45! and Jennings, H. J. and H. C. Lugowski, 1981.Immunochemistry of groups A, B and C meningococcalpolysaccharide-tetanus toxoid conjugates. J. Immunol. 127:1011-1018!).

Group B Meningococcus a bacteria group causing more than 50% of thecases of meningococcal disease in many countries is a group of bacteriawhose capsular polysaccharides do not induce immune reaction or inducelittle immune reaction. (Poolman et al., The Lancet, September 1986,pages 555-558).

Therefore, a search has been carried out for the group B Meningococcusfor the saccharide peptide conjugates that could be useful in a vaccineas capsular polysaccharides of this group do not give any immunereaction against gramnegative bacteria in test animals and humanvolunteers. Therefore, saccharide peptide conjugates were made withmodified capsular polysaccharide and carrier proteins ( Jennings, H. J.,R. Roy, and A. Gamian, 1986. Induction of meningococcal group Bpolysaccharide-specific IgG antibodies in mice using an N-proprionylatedB polysaccharide tetanus toxoid conjugate vaccine. J. Immunol.137:1708-1713! and Jennings, H. J. 1989. The capsular polysaccharide ofgroup B Neisseria meningitidis as a vehicle for vaccine development. InCruse, J. M., and R. E. Lewis, Conjugate Vaccines. Contrib. Microbiol.Immunol. Basel, Krager, 10:151-165!).

As it is suspected that anti group B antibodies (in particular IgG)demonstrate in vivo cross reaction with host antigens and as the use ofa saccharide peptide conjugate comprising modified capsularpolysaccharide of group B Meningococcus could therefore lead to causingauto immune disease most research is directed at a vaccine against groupB Meningococcus is directed at the potential use of sub capsularcomponents such as outer membrane proteins (OMP) and lipopolysaccharides(LPS).

So Jennings et al. describe conjugates of tetanus toxoid (TT) withdephosphorylated oligosaccharides (OS*) derived from meningococcal LPS.The immunogenicity of these OS*-TT conjugates was examined in rabbits(Infect. Immun. 43:407-412). Herein phosphoethanolamine was removed fromthe meningococcal oligosaccharides (OS) by treatment with hydrogenfluoride. The dephosphorylated oligosaccharides were subsequentlycoupled to tetanus toxoid. The thus obtained immunotype L₃ OS proteinconjugate was only slightly immunogenic in rabbits which can probably beexplained as a result of removal of the PEA group s.

In Infection and Immunity, March 1991, pages 843-851 Verheul et al.describe the preparation of meningococcal OS-protein conjugates withtetanus toxoid, wherein the phosphoethanolamine groups of theoligosaccharides have been maintained.

Oligosaccharides peptide conjugates, wherein saccharide portion andpeptide portion originate from different organisms (mostly a tetanus ordiptheria peptide) have the disdadvantage that oversensitivity ortolerance for said peptide portion (tetanus or diptheria carrier) canoccur and can therefore lead to reduced response to the B cellactivating part. Therefore, saccharide peptide conjugates provided withhomologic carrier peptide have been searched for. Such a saccharidepeptide conjugate will not only activate B cell memory but alsoreactivate T cell memory upon contact with the micro-organism inquestion.

Saccharide peptide conjugates are known comprising a homologous carrierpeptide. Paton et al, describe conjugation of pneumolysine toxoid totype 19F capsular polysaccharide of Streptococcus pneumoniae (Infectionand Immunity, July 1991, pages 2297-2304).

Saccharide peptide conjugates are also known comprising a homologouscarrier peptide comprising a saccharide part derived from alipopolysaccharide (LPS) of gram negative bacteria as immunity providingB cell activating part. Such a saccharide peptide conjugate offers asadvantage the possibility to make a vaccine providing immunity againstgramnegative bacteria, whose capsular polysaccharides provide no orinsufficient immune reaction. A saccharide peptide conjugate comprisinglipopolysaccharide as immunity providing B cell activating part also,however, has disadvantages. Lipopolysaccharide contains toxic parts anda saccharide peptide conjugate comprising a lipopolysaccharide withtoxic parts will also be toxic.

In Contrib. Microbiol. Immunol. Basel, Karger, 1989, vol. 10, pages166-189 Cryz. J. C. et al, describe vaccines against Pseudomonasaeruginosa comprising a saccharide peptide conjugate comprisingdetoxified lipopolysaccharide of Pseudomonas aeruginosa immunotype 5which lipopolysaccharide is coupled to various carrier proteins. Thecarrier proteins can be both homologous and non-homologous. The carrierproteins mentioned in this article are tetanus toxoid, toxin A and piliof Pseudomonas aeruginosa.

The LPS of Pseudomonas aeruginosa is made harmless according to thisarticle by removing ester bound fatty acids of Lipid A, so that theserologically active O polysaccharide portion can be incorporated in asaccharide peptide conjugate. Such detoxified LPA (D-LPS) was convertedto an active ester by coupling to N-hydroxysuccinimide and subsequentlythe active ester was coupled to a protein which had been provided with aspacer (1,4-diaminobutane) in order to simplify the coupling. TheD-LPS-TA-conjugate was not immunogenic however the D-LPS-pili-conjugateand the D-LPS-TT-conjugate were.

In an article by Boons et al. (Bioorganic and Medicinal ChemistryLetters, Vol. 1, No. 6, pages 303-308, 1991) it is described that asaccharide peptide conjugate (SPC) with two essential immunologicaldomains (i.e. the B and T epitopes responsible for antibody specificityand T helper activity) which are covalently bound by means of anartificial spacer has been made as part of vaccine development with abroad action against N. meningitidis. In this known conjugate the Bepitope function is provided by a fragment of the IC region of theLPS-immunotype 6 (IC=inner core). A T cell epitope comprising peptide isselected for inducing homologous T helper cell response (memory). The Tcell epitope comprising peptide part of the known homologous SPC is the47-59 part of a meningococcal OMP described by Wiertz E. J. H. J. et al(in Proceedings of the Eleventh American Peptide symposium, ed. River,J. E.; Marshall, G. R.; ESCOM, Leiden, 1990). Disadvantages of thisknown SPC are the necessary complicated and costly chemical synthesis onthe one hand hindering production on a large scale for economic reasonsand on the other hand the fact that only a part of the immuno action ofOMP is used because only a small fragment of OMP comprising a T cellactivating part is used, in respect of both B and T cell activatingparts.

Until now it appeared impossible to couple a saccharide part to acomplete OMP or a large OMP fragment comprising more than one epitopewithout changing the structure or composition which would lower theimmuno activity of the OMP.

Furthermore, it is known that microorganisms producing OMP's incorporatethese in their membrane in a certain configuration wherein portion ofthe OMP are anchored in the membrane and portions protrude in the shapeof loops. The specific configuration is required for the immuo activityof the T cell activating parts present in the OMP. In NIPH ANNALS,Volume 14, number 2, December 1991 it is described by Frederiksen J. H.et al on pages 67-79 in particular on 69-71, that microorganisms can betreated in such a manner that they "bleb", i.e. form outer membranevesicles (OMV's). Such outer membrane vesicles are provided with a verylarge number of OMP's on their surface and offer the possibility to beused as potent vaccine component as a large amount of OMP's are presentin the conformation required for immuno activity. However, a number ofthe disadvantages of other vaccines comprising solely OMP a immunoactivity activating component, are also valid for such a vaccinecomponent. Known OMV vaccines appear to be able to provide intermediateprotection in humans i.e. 50-80% protection.

The subject invention attempts to solve the problems that exist for thevarious known vaccine components. Firstly, the search has been directedat finding a B cell activating molecule that is small and simple toobtain, is not toxic and can also induce immunity against more than oneLPS immuno type. The subject invention is also directed at an immunityproviding B cell activating molecule derived from a meningococcallipopolysaccharide (LPS), said molecule comprising at least one epitope,said molecule comprising at least the common part of the oligosaccharidepart (core region) of lipopolysaccharides which are specific for atleast two meningococcal immunotypes, preferably immunotypes L2 and L3and wherein galactose is absent in the B cell activating part, as wellas at derivatives of the molecule with immune reaction inducingcapacity.

A suitable example of such a molecule is a molecule derived from the L2core of a meningococcal LPS and having the following structure: ##STR1##

Antibodies can be induced against such a structure which arespecifically bactericidal for immunotypes L2 and L3, the most prevelentimmunotypes. Another suitable example of such a molecule is a moleculederived from the L3 core and having the following structure: ##STR2##

This structure is also useful for inducing immune reaction against bothimmunotypes L2 and L3. Surprisingly, it has been found that an antibodywhich was known to exhibit cross reactivity for various meningococcalimmunotypes, but about which it was unknown what structure it recognizedcan bind the two molecular structures as indicated above. The twomolecular structures as indicated above differ from the epitopespostulated until now for L2 and L3 through the absence of galactose. Theepitopes postulated to date are not completely necessary for inducingbactericidal antibodies with cross reactivity for various meningococcalimmunotypes. The antibody used to illustrate this is mN8D6A and isdescribed in Infection and Immunity, October 1988, pages 2631-2638 (byKim, J. J., Mandrell, R. E., Zhen, H., Westerink, M. A. J., Poolman, J.T. and Griffiss, J. M.). In this article it is indicated that theantibody could bind to LPS to 28 group A N.meningiditis strains in dotblots and to multiple LOS components of various molecular weightsobtained from the 28 strains in immunoblots.

Molecules according to the invention can form a part of saccharidepeptide conjugates in the usual manner, wherein for example they arecoupled to tetanus toxoid or to another known suitable carrier invaccine technology. Advantageously a saccharide peptide conjugatecomprising a molecule according to the invention also comprises apeptide part with at least one T helper cell activating epitope, whichpeptide part preferably comprises at least one homologous protein or apeptide fragment derived from a homologous protein, wherein homologousmeans that both B cell and T helper cell activating epitopes are derivedfrom the same microorganisme. An outer membrane vesicle to which asaccharide peptide conjugate comprising a molecule according to theinvention has been added or has been incorporated as B cell activatingpart is a suitable embodiment, said embodiment can be usedadvantageously in a vaccine. Preferably, an outer membrane vesicle towhich a molecule or a saccharide peptide conjugate comprising such amolecule has been added, or in which such a molecule has beenincorporated comprises class I OMP or class I OMP fragments as T helpercell activating part.

The subject invention is also directed at methods for preparing suchlipopolysaccharide-derivatives according to the invention. Herefore, itis possible to apply mutagenesis, recombinant DNA techniques, enzymaticsplitting or chemical synthesis. The method via recombinant DNAtechniques comprises obtaining the molecule from a mutated or selectedproduction strain producing at least LPS without galactose. For this amutated meningococcal strain that does not produce galactose can beadvantageously applied. In particular, a production strain can beapplied that produces no or no functional galE anzym. In Proc. Natl.Acad. Sci. U.S.A. 86 (1989) pages 1669-1673 Frosch, M., Weisgerber, C.and Meyer, T. F. determined for N.meningitidis which part of theN.meningitidis genome comprises the genes for CPS production. This partwas cloned on plasmid pMF32.35 and we found that this plasmid alsocomprises sequences encoding LPS biosynthesis. A meningococcal strain inwhich this plasmide has been integrated was deposited on Jul. 29, 1993at CBS Baarn, the Netherlands, under number CBS 401.93. For an expert itis without a doubt via usual DNA techniques possible to apply one ormore mutations or deletions in this part of meningococcal DNA to preventgalactose production. Specifically it is possible to apply one or moremutations or deletions in the cps locus that is present on this plasmidthereby preventing expression of any or any functional GalE. With probescomprising a part of the sequence encoding a part of the enzym GalE thelocation of the GalE gene can be determined simply and subsequentlymutagenesis or deletion can be carried out in order to obtain a sequenceexpressing no or no functional GalE. The expression product of such amutated cps locus will produce lipopolysaccharide without galactose. Inthe usual manner an expression vector can subsequently be madecomprising such a mutated cps cassette or the relevant portion thereof.Now making a production strain with the aid of the mutated nucleotidesequence or the expression vector comprising such a sequence lies withinthe reach of an expert using typical protocols within the recombinantDNA technology. Advantageously a mutated meningococcal production strainin which a deletion in the wild type cps cassette has been made andwhich has been exchanged with a mutated cps cassette can be made in asimple manner.

Furthermore, the invention is directed at a solution to the problem ofcoupling OMP with a second component with immuno activity withoutdamaging the immuno activity of the OMP, so that a vaccine component canbe obtained comprising more than one T helper cell activating part. Thesubject invention is therefore directed at an outer membrane vesicleprovided with polypeptides having at least the immuno activity of outermembrane proteins (OMP's) bound to a membrane, said outer membranevesicle being characterized by the fact that the polypeptides are OMP'sor OMP fragments anchored in the membrane with a mutation in one of thesurface loops of the OMP from which the polypeptide has been derived.The invention is preferably directed at such an outer membrane vesicle,wherein the polypeptides are OMP's or OMP fragments anchored in themembrane with a mutation in one of the surface loops of the class 1 OMPfrom which the polypeptide has been derived. In particular, theinvention is directed at such an outer membrane vesicle wherein thepolypeptide comprises at least one mutation in one of the loops 2, 3, 5,6, 7 or 8.

It has been found that applying mutations in these loops does not reducethe immuno activity of membrane bound class-1-OMP. Thereby thepossibility to provide mutations at these locations enabling specificcoupling of the OMP to another desired component, preferably a componentwith additional immuno activity at the position of the mutation isoffered. An outer membrane vesicle according to the invention can beregarded as an outer membrane vesicle which has been activated forcoupling.

The mutation is preferably located in one of the loops 6 or 7. Theseloops are located in the tertiary structure of the OMP at the mostsuitable positions with regard to loops 1 and 4, said loops comprising anumber of important OMP epitopes.

The mutation can consist of at least the presence of an additional aminoacid with a reactive side chain in one of the loops 2, 3, 5, 6, 7 or 8.This can be an insertion, deletion or substitution in one of the loops.A substitution is preferred as the natural situation is than mostclosely imitated. The form the mutation takes is not critical, howeverthe location is.

As no cysteine is present in natural OMP and the sulfhydryl group is agood reactive group, this amino acid is preferably incorporated. Otheramino acids such as lysine are possible, however the presence of suchamino acids in the native OMP offers a number of reaction positions inthe OMP for coupling and therefore the resulting SPV's will have to bescreened in order to determine which have undergone the conjugation atthe desired location. Incorporating protective groups will is most caseslead to complicated chemical reactions with a large risk of denaturationof the polypeptide.

As indicated the polypeptide can comprise complete OMP but also an OMPfragment. The OMP fragment should possess sufficient length and astructure such that it can be anchored in the membrane of a microorganism and possesses at least immuno activating activity, preferablyas much as or more than the corresponding native OMP from which it hasbeen derived. An OMV according to the invention which is preferredcomprises a molecule according to the invention and/or a saccharidepeptide conjugate comprising a molecule according to the invention.

An application of an OMV according to the invention is in production ofa vaccine component that also comprises a conjugated saccharide part.The product of coupling a saccharide part to a polypeptide part of theouter membrane vesicle activated for coupling, an outer membrane vesicle(OMV) comprising a saccharide peptide conjugate (SPC), a so calledsaccharide peptide vesicle (SPV) will exhibit a broader immuno activitythan the existing outer membrane vesicles comprising OMP. The SPV willtherefore possess a larger and broader immuno activity then the knownSPC's. A further advantage is that the natural activity by which a microorganism anchors OMP's in the membrane can in an economical and simplefashion be used to produce a vaccine component with broader and strongerimmuno activity. The subject invention is directed at an SPV such as hasbeen described.

The coupling of a peptide part of an activated outer membrane vesicle toa saccharide part, said saccharide part comprising at least an immunityproviding B cell activating part with at least one epitope derived froma lipopolysaccharide (LPS) of a gramnegative bacteria and an outermembrane vesicle comprising a fragment of a B cell activating part withat least one epitope isolated from a gramnegative bacteria is preferred.The invention also comprises a SPV provided with polypeptides having atleast the immuno activity of OMP, said polypeptides forming a part ofsaccharide peptide conjugates, wherein the saccharide part and thepeptide part of such a conjugate are coupled at the location of themutation in one of the loops 2, 3, 4, 5, 6, 7 or 8 of the polypeptide.

A SPV comprising the LPS "core region" or a fragment derived therefromas B cell activating part of the saccharide part will be safer ascomponent of a vaccine than a SPV comprising a saccharide peptideconjugate comprising native LPS due to the absence of the toxic parts.Moreover, such a "core" containing saccharide peptide conjugate has theadvantage that it can contain a large number of different LPS "core" Bcell activating parts than a SPC with native LPS that becomes toxic,when too much of the toxic component lipid A is incorporated.

Meningococcal lipopolysaccharide is toxic and comprises three parts. InFIG. 1 a meningococcal LPS is illustrated. The lipid A part is toxic andthe lacto-N-neotetraose unit can perhaps lead to induction of autoantibodies. The oligosaccharide part of meningococcal LPS, that socalled "core region" is not toxic. The so called "inner core region" isthe part of the "core region" of the oligosaccharide part ofmeningococcal LPS in which the lacto-N-neotetraose unit is absent. Asaccharide peptide conjugate comprising a B cell activating part,comprising the "core" oligosaccharide of Meningococcus or a fragmentderived therefrom is a suitable example of a SPC that can be a part of aSPV according to the invention.

Indeed, a saccharide peptide conjugate comprising a saccharide partderived from Meningococcus lacking toxic lipid A and also lacking thecomplete lacto-N-neotetraose unit has been described by Boons et al,however, in this known vaccine component the saccharide part is coupledto a fragment derived from OMP comprising only one T cell epitope andthe SPC does not form a part of an outer membrane vesicle.

For a SPV according to the invention native LPS to be applied can beisolated and subsequently lipid A and a part of the tetraose unit can beremoved. Meningococcal lipopolysaccharide (LPS) can for example beisolated by the extraction method with hot water and phenol of Westphal(Westphal, O. and Jann. J. K., 1965, Methods Carbohydr. Chem. 5. 83-91).In short LPS is hydrolysed in 1% acetic acid unit flocculation occurs.Lipid A is removed by centrifugation and the oligosaccharides arepurified over a Biogel column. Subsequently, the major oligosaccharidecan be coupled to the peptide part.

"Core" oligosaccharide or an operative derivative thereof obtained bysuch a method can be incorporated in a saccharide peptide vesicleaccording to the invention.

As obtaining a B cell activating part derived from native LPS is atedious process and as complete purification of the "core"oligosaccharide is also problematic the B cell activating part of thesaccharide part of a saccharide peptide vesicle according to theinvention can be synthesized. A saccharide peptide vesicle according tothe invention can therefore comprises a synthetic B cell activating partin the saccharide part.

It is also possible to obtain a non toxic B cell activating part of theSPV derived from an LPS via a biochemical route, such as mutagenesis orenzymatic splitting. This route is possible if via molecular biologicalmethods a production strain is made that is capable of producing analtered lipid A and/or lacto-N-neotetraose. In any case, the terminalgalactose of the lacto-N-tetraose will have to be removed and a mutantproduction strain that does not produce galactose for incorporation inLPS can be made in a simple manner, as has been indicated for thepreparation of a molecule according to the invention above. Inparticular the subject invention is also directed at an SPV in thevarious embodiments that have already been extolled, wherein thesaccharide part comprises an immunity providing B cell activatingmolecule with at least one epitope derived from a meningococcallipopolysaccharide (LPS) said molecule comprising at least the communalpart of the oligosaccharide part (core region) of lipopolysaccharideswhich are specific for at least two meningococcal immunotypes preferablyimmunotypes L2 and L3 and wherein galactose is absent from the B cellactivating part, as well as derivatives of the molecule having immunereaction inducing capacity.

The saccharide peptide vesicles according to the invention can beconjugated by a spacer. This provides the advantage that nointramolecular reactions occur between the reactive groups of thesaccharide part and the peptide part. Such reactions can namely lead toan altered tertiary structure of the saccharide part and/or peptide partresulting in deterioration of immuno activity.

A number of LPS immunotype specific epitopes owe their activity to thepresence of at least one phosphoethanolamine (PEA) group. Therefore, asaccharide peptide vesicle according to the invention is preferredwherein PEA group s of the B cell activating part of the saccharide partcomprise free amino groups. An example of such a saccharide peptidevesicle according to the invention comprising an immuno specific B cellactivating part in the saccharide part, is a saccharide peptide vesiclecomprising LPS of meningococcus with immunotype L₃ in the saccharidepart. Such a saccharide peptide vesicle comprising an immuno specificepitope of immunotype L₃ will preferably comprise a B cell activatingpart with PEA group s having free amino groups.

Saccharide peptide vesicles according to the invention advantageouslycomprise a saccharide part comprising a B cell activating part that cangive cross section with more than one immunotype. A vaccine comprisingsuch a saccharide peptide vesicle according to the invention wil provideprotection against more than one immunotype.

At the moment twelve different lipopolysaccharides are known formeningococcal strains (which corresponds to 12 immunotypes). Thedifferences in the meningococcal LPS immunotypes have been localized inthe oligosaccharide part of the LPS ("core region"). Recently thecomplete primary structures of the oligosaccharides for immunotypes L₁,_(L) 2, L₃, L₅ and L₆ were postulated; these are illustrated in FIG. 2(Difabio J. L. et al. 1990, Structure of the L1 and L6 coreoligosaccharide of Neisseria meningitidis, Can. J. Chem. 86:1029-1034;Jennings, H. J. et al, 1987, Structure and Immunochemistry ofmeningococcal lipopolysaccharides, Anthonie van Leeuwenhoek 53:519-522;Michon, F. et al, 1990, Structure of the L5 lipopolysaccharide coreoligosaccharide of Neisseria meningitidis, J. Biol. Chem. 256:7243-7247;Verhuel, A. F. M. et al Infection and Immunity (1991), 51: p 3566-3573).

A saccharide peptide vesicle according to the invention comprising oneor more oligosaccharides is useful as component of a vaccine directedagainst at least one meningococcal immunotype. A saccharide peptidevesicle according to the invention according at least the B cellactivating part of at least one of the oligosaccharides in saidsaccharide part can therefore advantageously be used as component of avaccine directed against at least one meningococcal immunotype.

The oligosaccharides of the immunotypes differ with regard tomonosaccharide composition, amount and location of phosphoethanolamine(PEA) groups and the degree of acetylation of the α(1→2) boundGlCNAc-unit or other units. For most immunotypes the basic structure ofthe oligosaccharide "core" is the same.

As the "core" oligosaccharides of meningococcal LPS exhibit conformityfor various immunotypes the "core" comprises more than one immunotypespecific epitope. A SPV according to the invention that comprises such a"core" as B cell activating part of the saccharide part will bepreferred. Such a SPV according to the invention can have a simultaneousB cell activating effect on a number of different immunotypes. Asaccharide peptide vesicle according to the invention that can have asimultaneous B cell activating effect for a number of immunotypes canalso comprise a derived fragment comprising more than one immunotypespecific epitope.

It is also possible to incorporate only a relevant portion of the "core"oligosaccharide as fragment in a SPV according to the invention,providing specific immunity for specific immunotype. It is also possibleto incorporate more than one B cell activating part in the saccharidepart of a SPV according to the invention so that such a SPV comprisesvarious specific immunity providing parts and can therefore provideimmunity for various specific immunotypes.

B cell activating parts of the saccharide part with specific immunityproviding action against various immunotypes or B cell activating partsof the saccharide part having cross reactive immunity providing actioncan advantageously be incorporated in the saccharide part of asaccharide peptide vesicle according to the invention. It has becomepossible to synthesize well defined oligosaccharides with increasingcomplexity and molecular weight, so that oligosaccharides comprising oneor more B cell activating structures can be synthesized.

It is known about Meningococcus that immunotypes L3 and L2 cause approx.70% and 30% respectively of group B Meningococcal meningitis. Therefore,saccharide peptide vesicles according to the invention comprising atleast B cell activating parts of L3 and/or L2 immunotypes in thesaccharide part of the saccharide peptide vesicle are preferred.

A saccharide peptide vesicle according to the invention comprising atleast a B cell activating part of the saccharide part that is aderivative of ##STR3##

A saccharide peptide vesicle that is preferred is a SPV comprising a Bcell activating part in the saccharide part exhibiting at least crossreaction with two immunotypes of gram negative bacteria. An example ofsuch a saccharide peptide vesicle exhibits at least cross reaction withmeningococcal immunotypes L2 and L3. Such a saccharide peptide vesicletherefore comprises a molecule with structure ##STR4##

A saccharide peptide vesicle that is also preferred is a SPV comprisinga B cell activating part in the saccharide part exhibiting at leastcross reaction with meningococcus immunotypes L1 and L3.

A saccharide peptide vesicle which exhibits cross reaction with at leastimmunotypes L₁, L₂ and L₃ comprises at least the branchedoligosaccharide β-D-Glcp(1→4)- L-α-D-Hepp(1→3)!-L-α-D-Hepp) as B cellactivating part in the saccharide part. A suitable example of such asaccharide peptide vesicle according to the invention also comprises aPEA group on the 0-3 of the heptosylunit of the branched oligosaccharideβ-D-Glcp(1→4)- L-α-D-Hepp(1→3)!-_(L-) α-D-Hepp).

A saccharide peptide vesicle according to the invention exhibiting crossreaction with more immunotypes than L₁, L₂ and L₃ comprises at least thebranched oligosaccharide β-D-Glcp(1→4)- L-α-D-Hepp(1→3)!-L-α-Hepp)provided with a spacer giving a link with at least one anotherimmunotype specific B cell activating part with at least one epitope asB cell activating part in the saccharide part.

A SPV according to the invention can comprise a saccharide part that issynthesized and/or modified with regard to a natural B cell activatingpart as B cell activating part for a certain immunotype. Such asynthesized and/or modified B cell activating part will preferablyexhibit cross reaction with various immunotypes and/or give an improvedimmune reaction with regard to the corresponding part of the naturalLPS. In any case the terminal galactose should be removed, either viaenzymatic route, or via genetic manipulation, through mutation. As hasbeen indicated above the complete absence of galactose in suchstructures is a possible embodiment, which is obtainable by a personskilled in the art through recombinant DNA technology.

Such a synthesized and/or modified B cell activating part can arisethrough selective addition and/or deletion of certain groups and/orsugar units.

A saccharide peptide vesicle according to the invention will preferablycomprise an outer membrane proteine (OMP) or a fragment derived from anOMP as peptide part. Preferably, a SPV according to the invention willcomprise an OMP of Meningococcus class I as this OMP can induceprotective antibodies, does not possess a tendency to block antibodiesagainst other proteins and does not exhibit too great an antigenicvariation. Many recognition sites for human T cells have been researchedon the class I outer membrane protein of Meningococcus H44/76, making ita suitable starting strain for OMP to be used.

The subject patent application is also directed at a method forpreparing an outer membrane vesicle according to the invention. Thismethod comprises the following steps

1) a nucleotide sequence encoding the polypeptide is expressed in abacteria;

2) the bacteria is cultivated under known circumstances for producingouter membrane vesicles as is described in NIPH ANNALS, Volume 14,number 2, December 1991 by Fredriksen J. H. et al on p 67-79, inparticular on page 69-71 and the thus created membrane pieces withpolypeptide can optionally be isolated.

For preparing a SPV according to the invention subsequently

3) the membrane pieces formed in step 2 can be provided with saccharidepeptide conjugates by coupling the polypeptide to the desired saccharidepart.

For preparing an OMV or a SPV according to the invention the requirednucleotide sequence for step 1) to be used can be a recombinantnucleotide sequence encoding a class I OMP or a fragment thereof with atleast the immuno activity of class I OMP, in which in comparison to thecorresponding natural sequence a mutation has been created in one of thesurface loops. A mutation in one of the loops 2, 3, 4, 5, 6, 7 or 8 inparticular in loops 6 and 7 is preferred. The mutation results in acodon for at least one amino acid having a specific reactive side in oneof the surface loops. In particular the mutation results in a codon forcysteine. A saccharide part obtained by recombinant DNA technology orvia a synthetic route can be used in the method for preparing a SPVaccording to the invention. In particular a molecule according to theinvention which is obtainable as has been described herein elsewhere canbe used as saccharide part.

For coupling a saccharide part to an OMV according the invention inparticular to an OMV-SH provided with a cysteine group the followingreaction schedule can be followed: ##STR5##

More specifically the reaction can be executed as follows: BrCH₂--CO--NH--(CH₂)₄ --NH₂ (substance 1,excess)+OS-KDO+EDC+sulfo-NSH--^(pH8) OS-----CO--CH₂ Br (substance 2).Substance 2 is subjected to gel filtration and subsequently

    substance 2+OMV-SH→conjugate.

Another possibility for executing the coupling is with BrCH₂--CO--NH--(CH₂)₄ --NH--CO--CH₂ Br (substance 3, excess)+OS-SH (asdescribed on pages 34 and 35 of Andre Verhuel's thesis, MeningococcalLPS derived oligosaccharide protein conjugate vaccines Oct. 29, 1991,Utrecht). This results in OS-----CO--CH₂ Br (substance 2*). This issubjected to gelfiltration or optionally to a simple ET₂₀ -rinse.Substance 2* can subsequently be converted to the conjugate with OMV-SH.As substance 3 is lipophilic the modification of OS-SH occurs in amixture of an aqueous buffer and an organic solvent, such as dioxane.For this reason the reaction of OMV-SH with substance 3 followed afterpurification by incubation with OS-SH is less attractive.

For specific coupling of carbohydrates the presence of suitable groupssuch as an amino (--NH₂), carboxylic acid (--COOH), thiol (--SH) or analdehyde (CHO) is necessary within the carbohydrate antigen (Dick, W. E.et al., 1989, Glycoconjugates of bacterial carbohydrate antigens. InCruse, J. M. and R. E. Lewis, Conjugate vaccines, Contrib. Microbiol.Immunol., Basel, Krager, 10:48-114). These groups can be present in theantigen but must often be incorporated via chemical or enzymaticmethods. It is preferred to use a coupling method resulting in as smalla modification of the carbohydrate and protein antigen as possible. Theuse of a specific spacer to be incorporated is conducive for this. It ispreferred to destroy the epitope or epitopes present or to generateundesired immuno dominant neo antigens as little as possible. Theinfluence of the coupling method on the immunologic characteristics ofan oligosaccharide-peptide vesicle is large is small oligosaccharidesare used (Hoppner, W., et al., 1985, Study on the carbohydratespecificity of antibodies formed in rabbits to synthetic glycoproteinwith the carbohydrate structure of asialoglycophorin A. Mol. Immunol.12:1341-1348).

Most known vaccines are based on capsular polysaccharides or O antigensconsisting of repetitive units of 1 to 8 monosaccharides, in order tominimalise the influence of the coupling by use of largeroligosaccharides. Meningococcal LPS does not contain repetitive unitsand therefore the selection of the coupling method using meningococcalLPS will in particular be important for the immunologic andimmunochemical characteristics of the resulting conjugate.

Preferably the saccharide is coupled to the peptide via a spacercontaining the terminal reactive groups such as NH₂ and COOH. The use ofa spacer in saccharide peptide vesicles according to the invention hasthe advantage that the tertiary structure of the saccharide part is notaltered, which is important for the immuno activity of the saccharide.

In the meningococcal oligosaccharides two groups are available for usein coupling such an oligosaccharide to a carrier peptide: the free aminogroup of the phosphoethanolamine group (PEA group) and the carboxylicacid group of the KDO unit. The PEA group should preferably not be usedfor coupling as this group probably comprises part of a number of theimmunotype specific epitopes. Therefore, it is preferable in asaccharide peptide vesicle according to the invention to maintainphosphoethanolamine groups with free amino groups. Typically saccharidepeptide conjugates can be made on the basis of coupling the carboxylicacid group of the oligosaccharide to the free amino groups of thepeptide. When this method is applied with the meningococcaloligosaccharide this can result in coupling of oligosaccharide tooligosaccharide or of oligosaccharide to carboxylic acid groups of thepeptide by the PEA group of the oligosaccharide. Jennings et al.(Infect. Immun. 43:407-412) have solved this problem by removing the PEAgroups through treatment with hydrogen fluoride. The dephosphorylatedoligosaccharides were subsequently coupled to tetanus toxoid byincorporating β-(4-aminophenyl)ethylamine as spacer at the reducingterminus via reductive amination (which lead to loss of the ringstructure of KDO), followed by activation of the amino group withthiophosgen followed by coupling to tetanus toxoid. Such conjugates withimmunotype L₂, L₅ and _(L) 10 were very immunogenic in rabbits whereasthose of L₃ were badly immunogenic. For this last group the loss of PEAgroup s and/or ring structure of the KDO group appears to be importantfor the immunity and it is preferable to make a saccharide peptidevesicle with L₃ oligosaccharide of Meningococcus with the PEA group sand the KDO ring structure of the meningococcal oligosaccharide havebeen modified as little as possible.

The abovementioned methods do not suffice for preparing a saccharidepeptide vesicle according to the invention in which the B cellactivating activity of the saccharide part depends on the presence ofone or more phosphoethanol (PEA) groups. Additional measures arenecessary for preparing such saccharide peptide vesicles.

A possibility for preparation of a saccharide peptide vesicle accordingto the invention that solves the abovementioned problem is used of asaccharide part that has been synthetically obtained.

With current techniques it is possible to synthesize oligosaccharides.Furthermore, as has been described previously in this subject patternapplication, in the case of saccharide peptide vesicles comprisingoligosaccharides derived from LPS it can be advantageous to synthesizethe B cell activating part that is present in the "inner core" of LPS.Another advantage of a synthetically obtained saccharide part lies inthe possibility to incorporate the minimum saccharide that operates as Bcell activating part in the SPC according to the invention. Furthermore,it is also possible to synthesize a saccharide part comprising a minimumoligosaccharide with cross reactivity immunity providing activity. It isalso possible to synthesize B cell activating parts providing betterimmuno activity than the natural oligosaccharides.

It is simpler to place the spacer at a specific location with asynthetically obtained saccharide part. It is also possible toincorporate one or more phosphoethanol groups in the B cell activatingpart of the saccharide part for obtaining improved immuno activity.

In order to place the spacer at a specific location in the saccharide itis preferred to provide the reactive groups of the saccharide withprotective groups before the spacer is coupled. The reactive group atwhich the coupling with the spacer occurs is not provided with aprotective group. This method prevents groups which are essential forthe immunity providing activity to be removed or altered during couplingof the spacer.

The invention is also directed at a nucleotide sequence encodingrecombinant OMP or a recombinant OMP fragment which is discernible fromnon recombinant OMP or a non OMP fragment by at least a mutation in oneof the loops 2, 3, 6, 7 or 8 of the OMP. In particular the invention isdirected at such a nucleotide sequence with a mutation in one of theloops 6 or 7. A sequence also including the most important antigenicdeterminants located in loops 1 and 4 is preferred. The nucleotidesequence can comprise an insertion, deletion or alteration of at leastone codon as mutation, with a preference for a mutation leading to atleast formation of a codon including an additional amino acid with areactive side group in one of the loops 2, 3, 6, 7 or 8 in comparison tothe amino acid sequence of the corresponding OMP or OMP fragment. Thenucleotide sequence according to the invention further preferablycomprises a codon encoding cysteine.

The invention is also directed at an expression vector comprising thenucleotide sequence for OMP or an OMP fragment according to theinvention as well as a micro organism comprising said nucleotidesequence and/or said expression vector according to the invention.

The invention is also directed at a polypeptide having at least theimmuno activity of OMP, which polypeptide is characterized by thepresence of an additional amino acid with a reactive side chain in oneof the surface loops, in particular loops 2, 3, 5, 6, 7 or 8, incomparison with corresponding native OMP or a corresponding fragment ofthe native OMP. A polypeptide with an amino acid with a specificreactive side chain in loop 6 or 7 is preferred because these loops willbe of least hindrance to the tertiary structure of OMP of loops 1 and 4when a saccharide part is coupled. Preferably a polypeptide according tothe invention comprises a cysteine as amino acid. As native OMP does notcomprise cysteine it is in a polypeptide according to the inventionpossible to couple very specifically to cysteine. The incorporation ofan additional lysine is also one of the possibilities, however, it isnot preferred due to the presence of more than one lysine in native OMPand therefore also in the polypeptide. The coupling will thereforeprovide more than one conjugate and will require screening for thecorrect conjugate.

A saccharide peptide conjugate comprising at least one epitope of an OMPcharacterized by the presence of a polypeptide according to theinvention also falls within the scope of the invention, as does avaccine comprising an outer membrane vesicle, a SPV, a polypeptide or asaccharide peptide conjugate according to the invention in an effectiveamount.

Furthermore, the invention is directed at a nucleotide sequencecomprising meningococcal LPS encoding material, said nucleotide sequencebeing discernible from the wild type nucleotide sequence by at least onemutation or deletion whereby the expression product of the mutatednucleotide sequence does not comprise any or does not comprisefunctional GalE in contrast to the nucleotide sequence of the wild type.Such a nucleotide sequence can comprise a mutation in the cps locus ofmeningococcal DNA or equivalent DNA derived therefrom, for example inDNA derived from N.meningiditis such as on plasmid pMF32.25 which hasbeen integrated in meningococcal strain CBS 401.93 and deposited at theCBS in Baarn on Jul. 29, 1993. Preferably such a sequence comprises amutation in the D or E region. An expression vector comprising anucleotide sequence of LPS meningococcal encoding material according tothe invention, as well as a microorganism comprising such a nucleotidesequence and capable of expressing such a sequence fall within the scopeof the subject invention. In particular, a mutagenised meningococcalstrain that produces LPS without containing galactose falls within thescope of the subject invention as does a vaccine comprising such a microorganism or molecule according to the invention.

EXAMPLE I Summary

Class 1 outer membrane proteins are used for vaccine development againstB Meningoccocci. These proteins naturally contain two variable areas(=epitopes), which bind bactericidal antibodies. These epitopes arelocated in two loops 1 and 4 that are exposed at the surface. Fourmeningococcal strains were made with extra epitopes in loops 5 and 6 ofclass 1 protein (see table A), from which it is apparent that insertionat loops 5 and 6 is possible without disadvantages to epitopes in 1 and4. The strains all have the natural epitopes of the parent strainA8-6^(t) in loops 1 and 4, namely the P1.5 and P1.2 epitopes.Furthermore, strain J007 has an extra P1.7 and strain J016 has an extraP1.16 epitope, whereas strain J716 has two extra epitopes, the P1.7 andthe P1.16 in loop 6. Finally, strain P106 has an extra P1.16 epitope inloop 5. All these strains are capable of binding monoclonal antibodiesto the cell surface.

                  TABLE A                                                         ______________________________________                                        meningococcal                                                                 string    loop 1   loop 4    loop 5 loop 6                                    ______________________________________                                        J007      P1.5     P1.2             P1.7                                      J016      P1.5     P1.2             P1.16                                     J716      P1.5     P1.2             P1.7 + P1.16                              P016      P1.5     P1.2      P1.16                                            ______________________________________                                    

With the aid of this information new meningococcal strains can beproduced containing mutations in OMP which strains can be used toproduce outer membrane vesicles (=OMV's) consisting of pieces of outermembrane and are under certain circumstances secreted by N.meningitidis. These pieces can subsequently be coupled to LPS. The OMV'scan serve as vaccine against B Meningococci.

1.1 The structure of the class 1 OMP

The class 1 OMP's are cation selective pore proteins 15!. The protein isapprox. 374 amino acids long and is preceeded by a signal peptide of 19amino acids 1!. A model for the topology of the protein in the outermembrane predicts eight loops exposed at the surface.

The transmembrane sequences have been maintained between the variousNeisseria porins and form a β sheet structure. The hydrophilic exposedloops at the surface exhibit large variation in length and sequence.When the different class 1 OMP's are compared, the sequences exhibit ahomology of 90%. The variation in the sequences appears to be located inthe first and the fourth loop. These are referred to as variable regionsor epitopes. Bacterial antibodies appeared to bind the epitopes in loops1 and 4, which corresponds to the fact that these loops are the longest.These two epitopes determine the subtype specificity of the class 1 OMP6!.

1.2 Incorporation of extra epitopes in the meningococcal class 1 OMP

When the different epitopes of the class 1 OMP's are compared, in total10 different subtype specific epitopes can be discerned.Oligonucleotides with "sticky ends" are synthesized for two epitopes,the P1.7 and P1.16. These oligonucleotides were subsequently placed inloop 5 and loop 6 of the class 1 OMP gene.

The incorporation of the P1.7 and P1.16 oligonucleotides in the class 1OMP gene provided new strains with extra epitopes in the class 1 OMP.These strains already comprise the P1.5 epitope in loop 1 and the P1.2epitope in loop 4. The newly incorporated epitopes, P1.7 and P1.16 havebeen placed in loop 5 or 6. The expression of the incorporated epitopesin the new strains has subsequently been observed with whole cell ELISAand compared to the expression of the parent strain. Subsequently theouter membrane complexes (OMC's) have been isolated from both the newstrains and the parent strain which are used for the immunisation ofmice.

1.3 Materials

The used DNA & plasmid:

The plasmid pTZ19R is a plasmid derived from Pharmacia, encoding fora.o. Amp-resistance (Ampr) and the lac Z'-gene. The used plasmid 2-2ΔSEis a derivative of pTZ19R. This plasmid contains a 1.9 kB insert with apart of the class 1 OMP gene from meningococcal strain 2996 (P1.5,2),from amino acid 100. This insert is located at the EcoRI site of theMultiple Cloning Site (=MCS) of pTZ19R. Through a deletion in theMultiple Cloning Site from the SalI site of the EcoRI site (where theinsert begins), the class 1 gene has come into the same reading frame asthe lac Z' derivative of pTZ19R. The class I OMP gene in this plasmidcontains a KpnI restriction site in loop 5.

Another used plasmid pPH204 is again a derivative of 2-2ΔSE.

The class 1 OMP gene in this plasmid contains a new KpnI restrictionsite, namely in loop 6 instead of loop 5. The original KpnI site in loop5 has been replaced by a BamHI restriction site and subsequently a KpnIsite has been made in loop 6 by PCR mutagenesis 17!.

The used enzymes:

The used enzymes are all derived from Boehringer Mannheim, together withthe supplied buffers.

KpnI (vol. act.: 10 U/μl), with incubation buffer L.

SpeI (vol. act.: 10 U/μl), with incubation buffer H.

Sna BI (vol. act.: 8 U/μl), with incubation buffer M.

T4 DNA ligase (vol. act.: 1 U/μl), with T4 DNA ligase buffer.

With corresponding incubation buffers:

M buffer (1×): 10 mM Tris-HCl; 10 mM MgCl₂ ; 50 mM NaCl; 1 mMdithioerythritol (DTE), pH 7.5 (at 37° C.)

L buffer (1×): 10 mM Tris-HCl; 10 mM MgCl₂ ; 1 mM dithioerythritol(DTE), pH 7.5 (at 37° C.)

H buffer (1×): 50 mM Tris-HCl; 10 mM MgCl₂ ; 100 mM NaCl; 1 mMdithioerythritol (DTE) pH 7.5 (at 37° C.)

T4 DNA ligase buffer (10×): 660 mM Tris-HCl; 50 mM MgCl₂ ; 50 mMdithioerythritol; 10 mM ATP; pH 7.5 (at 37° C.)

The used monoclonal antibodies directed at the epitopes and LPS-types:

                  TABLE B                                                         ______________________________________                                        monoclonal                                                                    or LPS-type   type, subtype                                                   ______________________________________                                        MN16C13F4     P1.2        P1 subtype specific                                 MN22.19A9     P1.5                                                            MN14C11.6     P1.7                                                            MN5C11G       P1.16       LPS immunotype                                      MN14F21.11    L1          specific                                            4A8B2         L3(7.9)                                                         4D1-B3        L3(7.9)                                                         MN3A8C        L5                                                              MN4C1B        L6(4.9)                                                         MNA11G        L11,12                                                          MN9C10D       L8,10,11,12                                                     3A12-E1       L3,7,8,9                                                        MN15A17F12    L9, 10                                                          MN15A8-1      Lipid A                                                         ______________________________________                                    

all monoclonals derived from RIVM.

The other materials are mentioned in the methods (1.4).

The used bacterial strains:

E. coliK12 (NM522: This bacteria strain is Hsd⁻ (=does not containrestriction modification system)

meningococcal strain H44/76 B⁻ (P1.7,1): This bacteria strain is acapsule deficient class 3 deficient mutant of H44/76 (P1.7,16).

meningococcal strain A8-6^(t) B⁻ (P1.5,2): This bacteria strain is acapsule deficient mutant H44/77 (P1.7,16), which has been transformedwith the class 1 protein of 2996 (P1.5,2) 17!.

1.4 Methods

1.4.1 Control of pPH204 through transformation to Meningococci.

The constructed plasmid pPH204 (P1.2) has been transformed tomeningococcal strain H44/76 B⁻ (P1.7,16). Here the P1.16 epitope in loop4 of H44/76 B⁻ was exchanged with the P1.2 epitope of plasmid pPH204.With the aid of colony blotting and immunoblotting (see further) P1.2⁺colonies were isolated providing the strain J072 after control withSDS-PAGE, Western- and immunoblotting, see also table B in Results(1.5).

1.4.2. Incorporation of extra epitopes in meningococcal class 1 OMP

1.4.2.1 Incorporation of oligonucleotides in the plasmids pPH204 and2-2ΔSE

The used class 1 OMP gene in plasmid pPH204 contains a restriction sitefor KpnI in the part encoding loop 6. An oligonucleotide with KpnIsticky ends which encodes the P1.7 epitope as indicated below and inpreparation & insertion of oligonucleotides (1.4.3) was placed herein.Through incorporation of the oligonucleotide the KpnI restriction sitedisappeared and a unique SpeI restriction site was created. This plasmidis named construct pJB007.

2.1 oligonucleotide of the P1.7 epitope with KpnI sticky ends (SEQ IDNOS:1-4) ##STR6##

    ______________________________________                                        restriction enzym:  restriction enzym:                                        ______________________________________                                        A/CTACT             GGTAC/C                                                   TGATC/A             C/CATGG                                                   SpeI                KpnI                                                      ______________________________________                                         Upon insertion of the oligonucleotide in the plasmid, KpnI site disappears     and a unique SpeI site appears:                                           

    ______________________________________                                        left (SEQ ID NO:5 and 6):                                                                       right (SEQ ID NOS:7 and 8):                                 ______________________________________                                        5'-GGTACG|AAC GGC-3'                                                                   5'-GTT ACT|AGTACC-3'                               3'-CCATGC|TTG CCG-5'                                                                   3'-CAA TGA|TCATGG-5'                               ______________________________________                                    

In the same manner an oligonucleotide with KpnI sticky ends was placedin loop 6 encoding the P1.16 epitope as indicated below and inpreparation & insertion of oligonucleotides (1.4.3). Throughincorporation of the oligonucleotide the KpnI restriction site alsodisappeared here, however a unique SnaBI restriction site was created.This plasmid is named construct pJB016.

2.2 oligonucleotide of the P1.16 epitope with KpnI sticky ends (SEQ IDNOS:9 and 10) ##STR7##

    ______________________________________                                        restriction enzyme: restriction enzyme:                                       ______________________________________                                        TAC/GTA             GGTAC/C                                                   ATG/CAT             C/CATGG                                                   SnaBI               KpnI                                                      ______________________________________                                         From insertion of the oligonucleotide in the plasmid, KpnI site disappears     and a unique SnaBI site appears:                                          

    ______________________________________                                        left (SEQ ID NOS:13 and 14):                                                                    right (SEQ ID NOS:15 and 16):                               ______________________________________                                        5'-GGTACG|TAT ACC-3'                                                                   5'-ACC TTG|AGTACC-3'                               3'-CCATGC|ATA TGG-5'                                                                   3'-TGG AAC|TCATGG-5'                               ______________________________________                                    

Subsequently an oligonucleotide with SpeI sticky ends behind theoligonucleotide P1.7 was placed in loop 6 in the construct pJB007,whereby the oligo encodes the P1.16 epitope as illustrated below. Herebythe SpeI restriction site disappeared through incorporation of theoligonucleotide. This plasmid is named construct pJB716.

2.3 oligonucleotide of the P1.16 epitope with SpeI sticky ends (SEQ IDNOS:17-20) ##STR8## restriction enzyme: A/CTAGT

TGATC/A

SpeI

By cutting the incorporated oligonucleotide of the P1.7 epitope withSpeI followed by an insertion with the oligonucleotide of the P1.16epitope (with SpeI sticky ends) the SpeI site disappears:

    ______________________________________                                        left (SEQ ID NOS:21 and 22):                                                                    right (SEQ ID NOS:23 and 24):                               ______________________________________                                        5'-ACTAGC|TAT ACC-3'                                                                   5'-ACC TTG|TCTAGT-3'                               3'-TGATCG|ATA TGG-5'                                                                   3'-TGG AAC|AGATCA-5'                               ______________________________________                                    

The used class 1 OMP gene in plasmid 2-2ΔSE contains a KpnIrestriction-site in the part encoding loop 5. A P1.16 oligonucleotidehas been placed therein. Through incorporation the KpnI restriction sitedisappeared. This construct is named pPH016.

                  TABLE C                                                         ______________________________________                                        construct      in loop 6                                                                              in loop 5                                             ______________________________________                                        pJB007         P1.7                                                           pJB016         P1.16                                                          pJB716         P1.7.16                                                        pPH016                  P1.16                                                 ______________________________________                                    

1.4.2.2 Transformation of constructs to E. coli

Subsequently transformation of the four constructs to E. coli K12 NN522(see furtheron) was carried out. The transformants were grown underselective pressure of 100 μg/ml Ampicilline. Plasmid was isolated fromthe colonies formed (see furtheron) and checked for presence of thecorrect constructs. Construct PJB007 was checked for the disappearedKpnI and the formed SpeI site. The construct pBJ016 was checked for thedisappeared KnpI and the formed SnaBI site and the construct pJB716 waschecked for the disappeared SpeI site. With the aid of SDS-PAGE, Westernblotting & immunoblotting (see furtheron) with the suitable monoclonalsit was observed whether the insertions of the P1.7 and P1.16 epitopewere successful therefore exhibiting expression and binding antibodies.

1.4.2.3 Transformation of constructs to meningococci

Subsequently the constructs (pJB007, pJB016, pJB716) were used totransform meningococcal strain A8-6^(t) B⁻ (P1.5,2), wherebyrecombination of the meningococcal chromosomal DNA and the constructoccurred. With the aid of colony blotting and immunoblotting (seefurtheron) P1.7⁺ and the P1.16⁺ colonies were isolated resulting in thedesired strains after checking with SDS-PAGE, Western & Immunoblotting,see also Table C in results.

1.4.2.4 Checking of new strains with whole cell Elisa

The degree in which the incorporated epitopes were expressed in thestrains J007, J016, J716, J072 and P016, was observed and thisexpression was compared to the expression of the parent strains.

1.4.3 Preparation & insertion of oligonucleotides

The complementary non phosphorylated oligonucleotides as indicatedabove, were synthesized on the Applied Biosystems 3814 DNA synthesizer.Hybridisation occured by adding the complementary oligonucleotides inequal concentrations, warming to 95° C. and subsequently cooling to roomtemperature. This led to oligo's with the correct sticky ends. Theplasmid was digested with the correct enzyme, after whicholigonucleotides were ligated with the aid of T4 ligates in the plasmido/n at 15° C. (in the ratio 200 ng oligo/I μg plasmid). The ligationmixture was subsequently warmed to 80° C. and slowly cooled to roomtemperature to enable incorporation of oligonucleotide. Throughincorporation of the oligo's in the plasmid the used restriction sitedisappeared. The ligation mixture was consequently cut with this enzyme,whereby linearizing the selfclosing products under the plasmids. Upontransformation to E. coli K12 NM522 (see furtheron) these linear pieceswere degraded, so that all transformants must have an insertion. Withthe aid of SDS PAGE, Western blotting & immunoblotting (see furtheron)with monoclonals against the new epitopes the transformants wereselected for the correct orientation of the incorporatedoligonucleotide.

1.4.4 Transformation to E. coli K12 NM522

Transformation of plasmid DNA to E. coli was carried out according to13!. For this an E. coli K12 NM522 was used which was grown in LuriaBroth medium (Trypton 10 g/l; yeast extract 5 g/l; NaCl 10 g/l, pH 7.0)at 37° C. After transformation the E. coli was plated on a solidnutrition medium consisting of LB-medium with 1.5% (m/v) agar (Noble,Difco). For selection for ampicillin resistance 100 μg/ml ampicillin(Sigma) was added to the medium, after which growth occurred at 30° C.in order to prevent satellite formation.

1.4.5 Plasmid isolation

Plasmid isolation from E. coli was carried out according to the alkalinelysis method 13!.

1.4.6 Agarose gel electrophoresis

Agarose gel electrophoresis was carried out according to 13!. For this1% (m/v) (Sigma No A-9539) for a normal gel and a 1.2% (m/v) low meltingagarose (Nusieve GTG, FMC Bioproducts) were applied for a preparativegel. TBE buffer (RIVM) 1× concentrated was applied as buffer for thesegels: 0.089M Tris, 0.089M boric acid, 0.002M ETDA (pH 8.3). Themolecular weight marker used here was the lambda HindIII digest(Boehringer Mannheim) with fragment sizes of 125, 564, 2027, 2322, 4361,6557, 9416, 23130 bp. A 30% (m/v) sucrose solution with 0.25% (m/v)bromophenol blue served as loading buffer (5×). The electrophoresis wascarried out at 10 V/cm. Afterwards these were coloured with 0.5 μg/mlethidiumbromide and photographed.

1.4.7 Transformation to Meningococci

A plate with Meningococci was cultivated o/n with 5% CO₂ and damp tissueat 37° C. The cells of the fully cultivated plate were subsequentlyresuspended in 10 ml Muller Hinton medium (RIVM) with 10 mM MgCl₂ of 37°C. The bacterial suspension was subsequently diluted 1:5 in MullerHinton medium, after which 1 μg/ml plasmid DNA was added. Afterincubation for 3 hours at 37° C., 10⁴ dilution in sterile PBS (with0.01M phosphate, buffered physiological salt pH 7.2; RIVM) was carriedout and plating on a gonococcal agar plate (* supplement 3) occurred.After overnight cultivation the correct transformants were selected withthe aid of colony blotting. See furtheron, at colony blotting.

1.4.8 Colony blotting

The cells of a fully covered meningococcal plate were transferred to a0.45 μm nitro cellulose membrane (Schleicher & Schuell). Subsequentlythe bacterial cells were put in PBS (phosphate buffered phys. saline, pH7.2; RIVM) with 0.1% Tween 80 (Polyoxyethylene sorbitan monooleate,Merck) and inactivated for 1 hour at 56° C. Afterwards the excess cellswere wiped off and the blot was treated as in immunoblotting (seefurtheron).

1.4.9 SDS PAGE

SDS PAGE was carried out according to 8!. The gel consisted of a 5%(m/vol.) acrylamide stacking gel and an 11% (m/vol.) acrylamideresolving gel. The used reference proteins (RIVM) had the sizes 14, 20,30, 43, 67 and 94 kD. All samples for the SDS PAGE consisted of completecells.

For the E. coli cells these were cultivated o/n at 37° C. in LB-mediumwith 100 μg/ml ampicillin, with and without 1 mM IPTG(=isopropyl-B-D-thiogalactopyranoside, Sigma). Subsequently 1.5 ml ofthe suspension was centrifuged off. The pellet was suspended in 70 μl H₂O to which 130 μl loading buffer (Tris/HCl 0.625M pH 6.8; 10% SDS; 50%glycerol; 0.01% bromophenol blue; B-mercapto-ethanol=2.4:4:2:1) wasadded. The mixture was subsequently incubated for 10 min. at 95° C.

For the meningococcal cells from a fully cultivated plate (o/n, with 5%CO₂ and damp tissue at 37° C.) were dissolved in 10 ml PBS (with 0.01Mphosphate, buffered physiological saline, pH 7.2; RIVM), inactivated for30 min. at 56° C. and centrifuged off (10 min. at 4000 rpm). The pelletwas suspended in 500 μl H₂ O. 170 μl loading buffer and 0 to 35 μl H₂ Owere added to 35 to 70 μl bacterial suspension, total volume of 200 μl.This mixture was warmed for 10 min. at 95° C. and put on gel.

The used electrophoresis buffer consisted of 50 mM Tris/HCl (pH 8.3);380 mM glycine; 0.1% SDS. The electrophoresis was carried out at aconstant current of 40 mA. After electrophoresis the gels were:

1. or coloured in 1 hour at 56° C. with Coomassie brilliant blue (0.1%(w/vol.) in 10% (vol./vol.) acetic acid and 30% (vol./vol.) methanol)and decoloured for at least 1 hour at 56° C. in a 10% (vol./vol.) aceticacid/5% (vol./vol.) methanol solution.

2. or blotted on a 0.2 μm nitro cellulose membrane (Schleicher &Schuell), see furtheron at Western blotting.

1.4.10 Western and immunoblotting

The proteins separated on SDS PAGE were transferred to a 0.2 μmnitrocellulose membrane (Schleicher & Schuell) with the aid of the AncosSemi Dry Electroblotter A, for 1 hour at 0.8 mA/cm² gel, with 25 mMTRIS/HCl (pH 8.3); 192 mM glycerine; 20% methanol; 0.0375% SDS as blotbuffer. Subsequently the blot was rinsed for a quarter of an hour in PBS(phosphate buffered physiological saline, pH 7.2) with 0.1% Tween 80(Merck). Afterwards the blots were rinsed for half an hour in PBS 0.1%Tween and 0.3% caseine (hydrolysate, N-Z-AmineA, ICN Biochemicals). Theblots were subsequently incubated for at least 1 hour in PBS/0.1%Tween/0.3% caseine with the monoclonal antibody against the epitope tobe detected. After incubation it was rinsed 3×10 min. with PBS/0.1%Tween and subsequently incubated for half an hour with protein Aperoxidase conjugate (diluted 1:10.000) 9!. After rinsing again for 3×10min. with PBS/0.1% Tween and 1× with H₂ O the substrate/hydrogenperoxide mixture: 30 ml substrate A (substr. A: phosphate/citratebuffer, 0.02M Na₂ HPO₄ and 0.01M citric acid 1:1, pH 5.0) with 10 mlsubstrate B (substr. B: 24 mg tetramethyl benzidine and 80 mg DONS(Dioctyl-sulfosuccinate) in 10 ml 96% ethanol) and 20 μl hydrogenperoxide 30% (Merck) were added. After a few minutes green/blue bandsbecame visible on the blots where antibodies bind the proteins.Afterwards the blots were rinsed 3× with H₂ O and photographed.

1.4.11 Whole cell Elisa

A plate with Meningococci was grown o/n with 5% CO₂ and damp tissue at37° C. The cells were subsequently resuspended in 5 ml PBS (phosphatebuffered phys. saline, pH 7.2; RIVM) and inactivated for 30 min. at 56°C. Subsequently the suspension was diluted to an OD₆₂₀ of 0.1.Microtitre plates (Titertak PVC microassay plates U bottom) were filledwith 100 μl suspension/well (coating). The suspension was dried o/n at37° C. on for 2 days at room temperature. The plates were rinsed 3× withPBS with 0.1% Tween (Merck) shortly before use. 100 μl Solution with themonoclonal-antibody-dilution in PBS/0.1% Tween/0.5% Protifar (Nutricia)was added per well and incubated for 1 hour at 37° C. The plates wererinsed 3× with PBS/0.1% Tween. Subsequently the wells were filled with100 μl protein A peroxidase conjugate (RIVM, 1:100000× dilution) or antiIgM* conjugate (RIVM, 1:2000× dilution in PBS/0.1% Tween/0.5% Protifar)suspension/well and incubated for 1 hour at 37° C. After rinsing 3× withPBS/0.1% Tween 100 μl substrate C (substr. C: 1 ml tetra methylbenzidine (6 mg/ml alcohol 96%) and 22 μl H₂ O₂ 30% (Merck) in 60 ml0.11M NaAc buffer) was added per well, after which incubation took placefor 10 minutes. The reaction was stopped by blocking with 100 μl 2M H₂SO₄ /well. The absorptions were read with an ELSIA reader (BiokineticsReader EL312e of Bio-Tek Instruments) at 450 nm.

1.4.12 OMP isolation

A plate with Meningococci was grown o/n at 37° C. with 5% CO₂ and damptissue. The cells were subsequently resuspended in 5 ml liquidmeningococcal medium (RIVM) and divided over two flasks with 200 ml ofmeningococcal medium (RIVM). These flasks were shaken o/n at 37° C. Thecells were inactivated 30 min at 56° C., after which they werecentrifuged at 10,000 rpm for 10 min. (Beckman model J-21B, rotorJA-14). The top liquid was discarded and 10 ml 0.01M Tris/HCl pH 8.0 wasadded to every pellet. After resuspending the pellets the suspensionswere joined in one 50 ml tube. The tube was placed in ice water andsubsequently subjected to 15 min. ultrasonic vibration (Branson Sonifier250, position 4, 50%). After which centrifugation was carried out for 30min. at 4000 rpm (Heraeus Christ Minifuge GL with permanent rotor). Thetop liquid was centrifuged for 1 hour at 30,000 rpm and 10° C. (SorvallARC-1 ultracentrifuge Oil Turbine Drive (OTD)-65, rotor 70 T_(i)).Subsequently the pellet was resuspended in 4 ml 1% sarcosyl in Tris/HClpH 8.0 and centrifuged for 15 min. at 5000 rpm (Heraeus minifuge). Thetop liquid was centrifuged for 1 hour at 30,000 rpm and 10° C. (Sorvallultracentrifuge, rotor T865.1). After pouring of the top liquid thepellet was dissolved in 2 ml 0.01M Tris/HCl pH 8.0. Subsequently theyield was checked by determining the protein content (with the BCA*Protein Assay Reagent of Pierce, protocol according to themanufacturer). After which the OMP's were brought to a protein contentof 1 mg/ml in Tris/buffer and filled out to 50 μl/tube. The purity isdetermined with the aid of an 11% SDS-PAA-gel and an ELISA.

1.5 Results

1.5.1 Control of pPH204 through transformation to Meningococci

In order to check whether the plasmid pPH204 remains intact, thisplasmid was transformed to meningococcal strain H44/76 B⁻. Here the P1.2epitope of plasmid pPH204 was exchanged with the P1.16 epitope of strainH44/76. Selection for the correct transformants took place by means ofcolony and immunoblotting, whereby P1.2⁺ colonies were isolated. Thecolonies provided the strain J072 after control with the aid ofSDS-PAGE, Western immunoblotting. This strain has the P1.7 epitope inloop 1 and the P1.2 in loop 4.

1.5.2 Insertion of P1.7 oligonucleotide in the KpnI site of pPh204

The oligonucleotide for the P1.7 epitope was placed in the KpnI site ofpPH204, after which the resulting construct pJB007 was transformed to E.coli K12 NM522. Plasmid was isolated from the resulting colonies. Theisolated plasmid material was checked for disappearance of the KpnIrestriction site and the presence of the new SpEI restriction site. Inall checked transformants the KpnI restriction site had disappeared andthe new SpeI restriction site was present. The transformants were placeon SDS PAGE. After Western immunoblotting with monoclonal antibodiesdirected against the P1.7 epitope, 3 transformants comprising theoligonucleotide in the correct orientation were selected.

1.5.3 Insertion of P1.16 oligonucleotide in the KpnI site of pPH204

The oligonucleotide for the P1.16 epitope was placed in the KpnI site ofpPH204 (construct pJB016) and subsequently transformed. Plasmids oftransformants were checked for the disappearance of KpnI site and thenew SnaBI site, see FIG. 10. Three transformants having the P1.16epitope in the correct orientation were selected with Westernimmunoblotting, see FIGS. 11 and 12, 13, 14, and 15.

1.5.4 Isolation of P1.16 oligonucleotide in the SpeI site of pJB007

The oligonucleotide for the P1.16 epitope was placed in the SpeI site ofPJB007 (construct pJB716) and transformed. Plasmids of transformantswere checked for the disappearance of SpeI site. 1 Transformant havingthe oligonucleotide in the correct orientation was selected with Westernimmunoblotting.

1.5.5 Insertion of P1.16 oligonucleotide in the KpnI site of 2-2ΔSE

The oligonucleotide for the P1.16 epitope was placed in the KpnI site ofplasmid 2-2ΔSE (construct pPH016) and transformed. Plasmids oftransformants were checked for the correct incorporation of theoligonucleotide (data not included) 17!.

1.5.6 Transformation of the constructs to Meningococci

Subsequently the four constructs pJB007, pJB016, pJB716 and pPH016 weretransformed to meningococcal strain A806^(t) B⁻ (P1.5.2). Selection forthe correct transformants occurred by means of colony andimmunoblotting, whereby P1.7⁺ and P1.16⁺ colonies were isolated. Thisresulted after checking the strains according to Table D.

                  TABLE D                                                         ______________________________________                                        constructed meningococcal strains                                             meningococcal                                                                             in      in        in    in                                        strain      loop 1  loop 4    loop 5                                                                              loop 6                                    ______________________________________                                        J007        P1.5    P1.2            P1.7                                      J016        P1.5    P1.2            P1.16                                     J716        P1.5    P1.2            P1.7.16                                   J072        P1.7    P1.2                                                      P016        P1.5    P1.2      P1.16                                           ______________________________________                                    

1.5.7 Whole cell ELISA

The expression of the incorporated epitopes in the new strains wasobserved and compared with the parent strains A8-6^(t) (P1.5.2) andH44/76 (P1.7.16) Table D.

1.6 Conclusion & discussion

In this series of tests four new meningococcal strains were made withextra epitopes in loops 5 and 6. Strains J007 and J016 respectivelycontain the 1.7 and the P1.16 epitope in loop 6, whereas J716 carriesboth the P1.7 and P1.16 epitopes. Strain P016 contains the P1.16 epitopeexclusively in loop 5. The whole cell ELISA carried out shows that themonoclonal antibodies directed against the incorporated epitopes bindwell to reasonably well to the whole cells in comparison to the parentstrains H44/76 and A8-6^(t). The ELISA shows that the new class 1 OMP'scan be transported completely antibodies would occur. When strain H44/76is compared to the four strains with extra epitopes in loops 5 and 6,the P1.7 epitope appears to bind equally well in all cases. It does notmatter whether the P1.7 epitope is located in loops 1, 5 or 6.

1.7 Literature for Example I

1! Barlow A. K., Heckels J. E. and Clarke I. N., The class 1 outermembrane protein of Neisseria meningitidis: gene sequence and structuralimmunological similarities to gonococcal porins, Molecular Biology 1989,3(2), p. 131-139.

2! Frasch E. F., Zollinger W. D. and Poolman J. T., Serotyping antigensof Neisseria meningitidis and a proposed scheme for designation ofserotypes, Reviews of infectious diseases, vol. 7, no. 4, July-August1985, p. 504-510.

3! Gotschlich E. C., Meningococcal meningitis, In: Bacterial Vaccins(Ed. Germanier, R.), Acedemice Press; Inc, 1984, ch.8, p. 237-255

4! Maiden M. C. J., Suker J., McKenna A. J., Bygraves J. A. and FeaversI. M., Comparison of the class 1 outer membrane proteins of eightserological reference strains of Neisseria meningitis, MolecularMicrobiology(1991),5(3), p. 727-736

5! Klugman K. P., Gotschlich E. C. and Blake M. S., Sequence of thestructural gene (rpmM) for the class 4 outer membrane protein ofNeisseria menigitidis, homology of the protein to gonococcal protein IIIand Escherichia coli Omp A and construction of meningococcal strainsthat lack class 4 protein, Infection and Immunity, July 1989, p.2066-2071.

6! Ley P. van der, Heckels J. E., Virji M., Hoogerhout P. and Poolman J.T., Topology of outer membrane porins in pathogenic Neisseria, in press.

7! Lifely M. R., Moreno C. and Lindon J. C., An intergrated molecularand immunological approach toward a meningococcal group B vaccine.Vaccine, vol. 5, March 1987, p. 11-26

8! Lugtenberg B., Meijers J., Peters R., Hoek P. van de, Alphen L. van,Electrophoretic resolution of the major outer membrane protein ofEscherichia coli K12 into four bands, FEBS letters, 1975, vol. 58, no.1,p.254-258.

9! Nakane P. K., Kawaoi A., Perosidase-labeled antibody. A new method ofconjugation, J. Histochem. and Cytochem.,1974, no. 22, p. 1084-1091.

10! Nester E. W. Evans Roberts C., Pearsall N. N. and McCarthy B. J.,Microbiology 2nd edition, Eastbourne, Sussex, Holt Rinehart and Winston,1978, p. 433, 443, 474, 540-543, 591.

11! Peltola H., Safary A., Kaythy H., Karanko V. and Andre F. E.,Evaluation of two tetravalent (ACYW-135) meningococcal vaccins ininfants and small children: a clinical study comparing immunogenity ofO-acetyl-negative and O- acetyl-positive grou C polysaccharides.

12! Poolman J. T., Marie S. and Zanen H. C., Variability of lowmolecular weight, heat modifiable outer membrane proteins of NeisseriaMeningitidis, Infection and Immunity, December 1980, p. 642-648

13! Sambrook J., Fritsch E. F. and Maniatis T., Molecular cloning: alaboratory manual, 2nd edition, 1989, Cold Spring Harbour Laboratory,Cold Spring Harbour, N.Y. p.1.25-1.28, 1.82-1.84, 6.3-6.16, 6.18-6.19.

14! Saukonen K., Leinonen M., Abdillahi H. and Poolman J. T.,Comparative evaluation of potentional components for group Bmeningococcal vaccine by passive protection in the infant rat and invitro bacterial assay, Vaccin, vol. 7, August 1989, p. 325-328.

15! Tomassen J., Vermeij P., Struyve M., Benz R. and Poolman J. T.,Isolation of Neisseria meningitidis mutants deficient in class 1 (PorA)and class 3 (PorB) outer membrane proteins, Infection and Immunity, May1990, p. 1355-1359.

16! Tsai C. M., Frasch C. E. and Mocca L. F., Five structural classes ofmajor outer membrane proteins in Neisseria meningitidis, J. Bacteriol.,1981, no. 146, p.46-78.

17! P. van der Ley personal information.

EXAMPLE II Summary

In this example the creation of a mutated outer membrane protein isdescribed, which OMP offers a coupling possibility for coupling theclass 1 OMP to the oligosaccharide part of the lipopolysaccharide. Forthis reason an oligonucleotide encoding a cysteine is incorporated in anexisting restriction site of the class 1 protein gene, said oligo issubsequently transformed to a capsule deficient mutant of meningococcalstrain H44/76, H44/76-B⁻. After transformation cysteine incorporationdid not appear to have any influence on epitope expression or anproduction of the class 1 OMP.

The selection of loops 5 and 6 was determined by the fact that theycontain no important class 1 epitopes and by the maintenance of immunoactivity illustrated in Example I after providing mutations in theseloops. Checking the result of incorporation took place on the basis ofepitope expression of the resulting transformants.

2.1 Material

The used plasmid:

The used plasmid, pPH204, is derived from the pTZ19R plasmid ofPharmacia. ##STR9## Used enzyms: KpnI (vol. act.: 10 U/μl); withincubation-buffer L.

PstI (vol. act.: 10 U/μl); with incubation-buffer H.

T4 DNA Ligase (vol.act.: 1 U/μl); with T4 DNA LIGASE buffer.

The composition of the corresponding incubation buffer, see Table E.

                  TABLE E                                                         ______________________________________                                        Composition of used buffers                                                   Buffer components               T4 DNA                                        in mmol/l    H           L      Ligase                                        ______________________________________                                        Tris-HCl     50          10     66                                            MgCl.sub.2   10          10     5                                             NaCl         100                                                              Dithioerytheritol                                                                          1           1      5                                             ATP                             1                                             pH at 37° C.                                                                        7.5         7.5    7.5                                           ______________________________________                                    

Both the enzymes and buffers are derived from BOEHRINGER MANNHEIM.

Used bacteria:

Escherichia coli K12 NM522: this strain does not comprise a restrictionmodification system.

Meningococcal strain: H44/76-B⁻. This is the mutation of H44/76 lackinga capsule.

Used monoclonals: see Table F.

                  TABLE F                                                         ______________________________________                                        Used monoclonals                                                              monoclonal    directed against                                                ______________________________________                                        MN16C13F4     P1.2                                                            MN14C11.6     P1.7                                                            MN5C11G        P1.16                                                          ______________________________________                                         All used monoclonals are derived from the RIVM.                          

Used membrane:

For Western Blot

BIO RAD Trans Blot Transfer medium pure Nitro cellulose membrane

Blotting Filter Paper, 0.45 micron.

Lot.No.: 4072/87020

Cat.No.: 162-0113

For Colony Blot

SCHLEICHER & SCHUELL BA 85/22, 0.45 micron.

diameter: 82 mm

Ref.No.: 406 216

2.2 Methods

2.2.1 Hybridisation of Oligonucleotides

The complementary nonphosphorylated oligonucleotides (OLIGO'S) (seebelow) were added in a concentration of 1 μg/100 μl each, and warmed to95° C. After warming the mixture was able to slowly cool down to roomtemperature. Beacause the cooling down occurs slowly hybridisation ofthe complementary oligonucleotides can occur 5!. In this manner oligo'swith the correct sticky ends are created, i.e. KpnI sticky ends.

Information C1-C2 Oligonucleotide

Oligonucleotide encoding cysteine with KnpI sticky ends (SEQ IDNOS:25-28)

    ______________________________________                                                     Gly     Cys   Ser   Leu   Ser                                    5'-    G     GGC     TGC   AGC   CTA   AGT   AC-3'                            3'-CATG                                                                              C     CCG     ACG   TCG   GAT   T                                      5'                                                                                         Ala     Ala   Ala   End                                          Restriction site for KpnI:                                                                          5'-GGTACC-3'                                                                3'-CCATGG-5'                                              Restriction site for PstI:                                                                          5'-CTGCAG-3'                                                                3'-GACGTC-5'                                              ______________________________________                                    

The oligonucleotides is composed of two single-strandedoligonucleotides:

    Oligo C1 (SEQ ID NO:29) 5'-GGG CTG CAG CCT AAG TAC-3'

    Oligo C2 (SEQ ID NO:30) 5'-TTA GGC TGC AGC CCG TAC-3'

2.2.2 Ligation of Hybridised Oligonucleotides in Plasmids

The plasmids were subjected to digestion with the correct enzyme andsubsequently separated on a 1.2% low melting gel from the plasmids thatwere not cut. Afterwards the gel was coloured with ethidium bromide andthe cut plasmids were cut out of the gel. After which the cut plasmidDNA was isolated from the agarose gel again through phenol extractionand ethanol precipitation 8!.

Half of the isolated plasmids were taken for ligation of the oligo's.The ligation mixture was composed as follows: 50 μl, cut plasmid DNA, 20μl hybridised oligo mixture, 10 μl ligation buffer, 4 μl T₄ ligase, 16μl dH₂ O. After incubation of the ligation mixture overnight (o/n) at16° C. the mixture was concentrated by ethanol precipitation.Subsequently the ligation products were separated on a 1.2% low meltinggel and the ligation products were cut out after which they wereisolated from the gel by phenol extraction and ethanol precipitation.

2.2.3 Melting Excess Oligo

After isolation of the ligation products from the gel these were warmedto 65° C. so the excess oligo was melted. By slowly cooling the mixturedown again hybridisation could occur between the complementary parts ofthe oligo ligated to the plasmid.

2.2.4 Removal of Selfclosing Plasmids

The solutions were subsequently postcut with the original restrictionenzyme in order for the selfclosing plasmids to be reopened. Thispostcutting could take place as the incorporation of the oligo hadchanged the original restriction site to a different restriction site.After cutting linear plasmids without oligo and circular plasmids withan incorporated oligonucleotide were created. As only circular plasmidDNA can be taken in by E. coli, in principle only transformantscontaining the oligo can arise.

2.2.5 Transformation to E. coli

Transformation procedure see: 8!.

After transformation the transformants were plated on Luria Broth (L.B.)medium nutrition media to which 100 μg/ml ampicillin was added. Theplates were incubated o/n at 37° C., after which the transformants wereplated from single colonies on L.B. nutrition media with ampicillin andincubated again o/n after which single colonies were transferred toliquid L.B. medium with ampicillin, which mixture was incubated o/n at37° C. whilst being shaken and was used for plasmid isolation (seefurtheron).

2.2.6 Plasmid Isolation

Plasmid isolation from the transformants was carried out according tothe alkaline lysis method 8!.

2.2.7 Digestion of the Recombinant-Plasmids

In order to check whether the oligonucleotide was present in theplasmids, a digestion was carried out with the restriction enzymeencoding the new restriction site. A digestion was also carried out withthe restriction enzyme encoding the restriction site which should havedisappeared, so that it could be determined on the basis of therestriction pattern on an agarose gel whether the old restriction sitehad disappeared and the new site was really present. The proteincomposition of the positive transformants was checked with sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS, PAGE).

2.2.8 SDS PAAGE

The SDS PAAGE was carried out according to protocol 8!. An 11%acrylamide gel was used as separation gel, a 5% acrylamide gel was usedas concentration gel.

Procedure with E. coli

The positive transformants were grown for electrophoresis o/n in liquidLB medium with (100 μg/ml) ampicillin both with and without (1 mM) IPTG{=isopropyl-8-D-thiogalactopyranoside (Sigma)}. 1.5 ml of the o/nculture was centrifuged off, after which the pellet was resuspended in40 μl dH₂ O, to which 10 μl loading buffer {250 mM Tris-HCl pH 6.8, 10%SDS, 10% dithiotreitol, 50% glycerol, 0.05% bromophenol blue} wereadded, after which the suspension was warmed for 10 minutes at 95° C.and put on gel.

Procedure with Meningococci

For the Meningococci the grown o/n in a damp atmosphere with 5% CO₂ on ameningococcal plate enriched with isovitalex were suspended in 10 ml PBS(0.01M phosphate buffered physiological saline, pH 7.2: RIVM). Theobtained suspension was subsequently warmed for 30 min. at 56° C., inorder for the Meningococci to be inactivated after which the suspensionwas centrifuged off (10 minutes at 4000 rpm). The pellet was suspendedin 40 μl dH₂ O and to which 10 μl loading buffer was again added afterwhich the suspension was warmed for 10 min. at 95° C. and put on gel.

The applied electrophoresis buffer had the following composition: 50 mMTris/HCl (pH 8.3), 380 mM glycine, 0.1% SDS. The electrophoresis wascarried out with 20 mA per gel.

After the electrophoresis the gel was:

1) coloured with Coomassie brilliant blue (1 hour at 56° C.), andafterwards coloured with 10% HAc/5 (vol./vol.)% methanol (3 times 30min. at 56° C.).

2) blotted on a 0.45 micron nitrocellulose membrane (Biorad).

2.2.9 Western or Immunoblotting

With the aid of an Ancos Semi Dry Electroblotter A the proteins of theSDS PAAgel were blotted over on to a 0.45 micron nitrocellulose membrane(Biorad). The protein transport from the gel to the membrane occurred byblotting for 1 hour at 0.8 mA/cm² gel whereby 25 mM Tris-HCl (pH 8.3),192 mM glycine, 20% methanol, 0.0375% SDS was used as blot buffer.

After blotting the blot was rinsed for a quarter of an hour in PBS with0.1% Tween 80 (polyoxyethylene sorbitan monooleate, Merck). Subsequentlythe blot was rinsed for half an hour with PBS to which 0.1% Tween 80 and0.3% caseine hydrolysate (N-Z Amine A, ICN Biochemicals) was added,after which incubation occurred for an hour with monoclonals dissolvedin PBS/0.1% Tween 80/0.3% caseine, against the epitope to be detected.After which the blot was rinsed 3 times for 10 min. with PBS/0.1% Tween80 after which it was incubated for half an hour with protein Aperoxidase conjugate, diluted 1:10.000 with PBS/0.1% Tween 80/0.3%caseine. Subsequently rinsing again took place 3 times 10 min. withPBS/0.1% Tween 80 and once with d^(H2) O after which the hydrogenperoxide substrate mix (20 μl 30% hydrogen peroxide, Merck; 30 mlsubstrate A:phosphate/citrate, 0.02M Na₂ HPO₄ and 0.01M citric acid(1:1) pH=5.0; 10 ml substrate B:80 mg Dioctyl-sulphosuccinate (DONS), 24mg tetra methyl benzidine (TMB) in 10 ml 96% ethanol) were added.

After several minutes blue bands became visible at the position wherethe monoclonal antibodies had bound to the corresponding proteins.Afterwards the blots were rinsed another 3 times with water after whichthey were photographed or were kept in the dark until they werephotographed.

2.2.10 Transformation to Meningococci

Meningococci were grown o/n on GC agar (Difco), enriched with isovitalexat 37° C. in a damp atmosphere with 5% CO₂ (3). the cells of thecompletely covered plate were resuspended in 10 ml Muller Hinton medium(RIVM), with 10 mM MgCl₂ of 37° C. Subsequently the suspension wasdiluted 1:5 in Muller Hinton medium with 10 mM MgCl₂ and 1 μg/ml plasmidDNA was added. Subsequently incubation for 3 hours at 37° C. took placeafter which the bacterial suspension was diluted 10⁴ times with sterilePBS and plated on a GC plate. After cultivation o/n the correcttransformants were selected by means of a colony blot.

2.2.11 Colony Blot

The colonies of a plate covered o/n at 37° C. in a damp atmosphere with5% CO₂ were blotted over on to a 0.45 micron nitrocellulose membrane(Schleicher & Schuell). Subsequently the blot was warmed for 30 min. inPBS/0.1% Tween 80 at 56° C. in order for the Meningococci to beinactivated. After warming the excess bacteria were wiped off. For thefurther procedure see the Western or immunoblot technique.

2.2.12 OMP Isolation

A plate with Meningococci was grown o/n at 37° C. in a damp atmospherewith 5% CO₂ after which the cells were resuspended in 5 ml meningococcalmedium. 200 ml Meningococcal medium was seeded with 2.5 ml of themixture. These 200 ml were activated o/n at 37° C., after which theMeningococci were inactivated by incubation during half an hour at 56°C. Subsequently the Meningococci were pelleted by centrifuging themedium for 10 min. at 10.000 rpm (centrifuge: centrikon T-324, rotorA6.9). The supernatant was poured off and the pellet was resuspended in10 ml 0.01M Tris/Hcl pH 8.0. Subsequently this solution wasultrasonically vibrated for 15 min. (Branson Sonifier 250, position 4,50%) whereby the solution was placed in an icebath. Subsequently thesonified solution was centrifuged at 10 min. at 5000 rpm (centrifuge:centrikon T-324, rotor A8.20). The supernatant was subsequentlycentrifuged for one hour at 20.000 rpm. and 10° C. (centrifuge:centrikon T-324, rotor A8.20). The pellet thus formed was resuspended in4 ml 1% sarcosyl in 0.01M Tris/HCl pH 8.0, after which centrifugationtook place for 5 min. at 5000 rpm. The supernatant was subsequentlycentrifuged for one hour at 20.000 rpm. and 10° C. (centrikon T-324centrifuge, rotor A8,20). The thus formed pellet was resuspended in 1 ml0.01M Tris/HCl pH 8.0. The yield was determined with the MicroassayProcedure (BIORAD). The purity of the OMP's was checked with SDS PAAGE.

2.2.13 Transformant Check with the Aid of Polymerase Chain Reaction

Day 1

The Meningococci for undergoing PCR were seeded from a GC plate,enriched with isovitalex, and cultivated o/n in a damp atmosphere with5% CO₂ at 37° C.

Day 2

A cell suspension was made of each of the strains to be subjected to PCRby resuspending a small flock of bacteria in 1 ml sterile distilledwater and subsequently warming this suspension for 10 min. at 95° C. Theresulting solution was subsequently shortly centrifuged and kept on ice.The reaction mixture was composed as follows:

10 μl buffer (500 mM KCl, 100 mM Tris/HCl pH 8.3, 15 mM MgCl₂, 0.1%{w/vol.} gelatine)

200 μM of each of the dNTP's

100 mg of each of the primers

25 μl cell suspension

filled to 100 μl with sterile distilled water.

100 μl mineral oil.

The PCR conditions were as follows: see Table G

                  TABLE G                                                         ______________________________________                                        PCR conditions                                                                Cycle number                                                                             min. 95° C.                                                                        min. 55° C.                                                                      min. 72° C.                           ______________________________________                                        1          5           1         2                                            2-30 incl. 1           1         2                                            ______________________________________                                    

After the 30th cycle the samples were held for a further 8 minutes at72° C. in order to make all the DNA present in the samplesdouble-stranded.

After PCR the samples were subjected to a phenol extraction and anethanol precipitation in order to obtain the produced DNA in pure form.

Day 3

10% of the purified DNA was electrophorated on a 0.1% agarose gel inorder to observe the result. From this it should be apparent whether theincorporated nucleotide was present in the DNA.

2.2. Result

2.2.1 Transformation of the Recombinant Plasmids to E.coli

The hybridised oligonucleotide was ligated in pPH204 cut with KpnI afterwhich the whole was transformed to E. coli K12 NM522. The plasmids wereisolated from the resulting transformants. The isolated plasmids werecut with KpnI and PstI and subsequently electrophorated on a 0.8%agarose gel.

                  TABLE H                                                         ______________________________________                                        Further illustration of FIG. 16                                               lane number                                                                              sample description                                                                           remark                                              ______________________________________                                        1,6,11,21,26                                                                             recombinant plasmid                                                                          uncut                                                          DNA                                                                2,4,7,9,12,14,                                                                           recombinant plasmid                                                                          KpnI site should                                    17,19,22,24,27                                                                           DNA KpnI digest                                                                              have disappeared                                    3,5,8,10,13,15,                                                                          recombinant plasmid                                                                          through incorporation                               18,20,23,25,28                                                                           DNA PstI digest                                                                              of the oligo a second                                                         PstI site has result-                                                         ed, now a fragment                                                            is cut out of the                                                             plasmid                                             16         Lambda marker  HindIII digest                                      ______________________________________                                    

From the formation of a fragment of approx. 600 base pairs from the PstIdigest and the disappearance of the KpnI site it is apparent that theoligo has been incorporated in pPH204.

The transformants were subsequently cultivated o/n at 37° C. in liquidL.B. medium with 1 mM IPTG and 100 μg/ml ampicillin. The bacterialproteins were subsequently separated with the aid of SDS PAGE afterwhich immunoblotting was used to see whether the oligo had beenincorporated in the correct orientation. Using the blotting the size ofthe class 1 protein was observed by demonstrating the P1.2 epitope withthe monoclonal MN16C13F4.

The transformants in the lanes No. 1, 3, 5, 7, 9, 11 show a protein bandwhich has travelled less far than those of the transformants in theremaining lanes. This indicates that the produced protein is larger thanin transformants in the lanes No. 2, 4, 6, 8, 10. The transformants inthe odd lanes comprise the oligonucleotide in the correct orientationand those of the even lanes in the incorrect orientation, therebycreating a stop codon resulting in a smaller protein.

2.3. Transformation to H44/76 B⁻

The plasmids of the positive transformants were subsequently transformedto H44/76 B⁻, a capsule deficient mutant of H44/76. The transformantswere selected by colony blotting for the P1.2 epitope with themonoclonal MN16C13F4. The outer membrane proteins of the puretransformants were isolated. The purity was checked by means of SDSPAAGE and immunoblotting. It was also checked whether the ratio betweenthe class 1 protein and the other proteins had changed with thetransformants. This appeared not to be the case (results not included).

Subsequently it was checked whether the P1.16 epitope had disappearedfrom the transformants, as a test the monoclonals against the P1.2 andthe P1.7 epitopes were included as well as strain H44/76 B⁻ as teststrain.

The monoclonal MN5C11G directed against the P1.16 epitope, only reactswith the parent strain. The monoclonal MN14C11.6 directed against theP1.7 epitope reacts with both the parent strain and the transformants.MN16C13F4 directed against the P1.2 epitope only reacts with thetransformants.

2.4 Conclusion

The successful introduction of a cysteine residue in the class 1 proteinof H44/76-B⁻ is apparent from the following results:

1) upon incorporation of the oligonucleotide the KpnI site hasdisappeared and a new PstI site has been formed.

2) after transformation to H44/76-B⁻ this has changed from P1.7.16 toPI.7,2 which is indicative of the fact that the gene encoding class 1protein present in plasmid pPH204 in which gene the oligo wasincorporated has been introduced into the Meningococcus. Cysteineincorporation does not appear to have an influence on the production ofthe class 1 protein considering the amount produced by theMeningococcus. The epitope expression by the bacteria also appears toremain normal.

2.5 Literature List for Example 2

1 FRASCH, C. E. 1977. Role of protein serotype antigens in protectionagainst disease due to Neisseria meningitidis. J.Infect.Dis. 136 (suppl)S84-S90.

2 FRASCH, C. E., W. D. ZOLLINGER and J. T. POOLMAN. 1985. Serotypeantigens of Neisseria meningitidis and a proposed scheme for designationof serotypes. Rev.Infect.Dis.7:504-510

3 GRIFFISS, J. M., B. L. BRANDT, D. D. BROUD, D. K. GOROFF AND C. J.BAKER. 1984. Immune response in infant children to disseminatedinfections with Neisseria meningitidis. J. Infect. Dis. 150:71-79.

4 KIM. J. J., R. E. MANDRELL, H. ZHEN, M. A. J. WESTERINK, J. T. POOLMANand J. M. GRIFFISS. 1988, Electromorphic characterization anddescription of conserved epitopes of the lipooligosaccharides of group ANeisseria meningitidis. Infect.Immun. 56:2631-2638.

4A KLUGMAN, K. P., GOTSCHLICH, E. C. and BLAKE, M. S. 1989. Sequence ofthe structural Gene (rmpM) for the class 4 outer Membrane Protein ofNeisseria meningitidis, Homology of the protein to Gonococcal ProteinIII and Esscherichia coli OmpA and Construction of meningococcal strainsthat lack Class 4 protein. Infect. and Immun., July 1989, P2066-2071.

5 LEY, P. van der, personal information.

5A MAIDEN, M. C. J., J. SUKER, A. J. McKENNA, J. A. BYGRAVES and I. M.FEAVERS. 1991. Comparison of the Class I outer membrane proteins ofeight serological reference strains of Neisseria meningitidis. Molec.Microbiol. 1991. 5:3:P727-736.

6 POOLMAN, J. T. 1990. Polysaccharides and Membrane vaccines, BacterialVaccines, P57-86 (70).

7 POOLMAN, J. T., C. T. P. HOPMAN, and H. C. ZANEN, 1982. Problems inthe defenition of meningococcal serotypes. FEMs Microbiol. Lett.13:339-348.

8 SAMBROOK, FRITSCH, MANIATIS. Molecular Cloning, a laboratorium manual,second edition, Cold Spring Harbour Laboratory, N.Y. P1.25-1.28,P1.82-1.85, P6.3-6.16, P6.18-6.19, P18.47-18.55.

9 SAUKONEN, K., M. LEINONEN, H. ABDILLAHI and J. T. POOLMAN, 1989.Comparative evaluation of components for group B meningococcal vaccinebij passive protection in the infant rat and in vitro bactericidalassay. Vaccine 7:325-328.

10 TSAI, C. M., C. E. FRASCH AND L. F. MOCCA. 1981. Five structuralclasses of major outer membrane proteins in Neisseria meningitidis.J.Bacteriol. 146:89-78.

11 VEDROS, N. A. 1987. Development of meningococcal serogroeps. p.33-38N. A. Vedros (ed.). Evolution of meningococcal disease, vol.2. CrC PressInc., Boca Raton, Fla.

12 ZOLLINGER, W. D., and R. E. MANDRELL. 1977. Outer membrane proteinand lipopolysaccharide serotyping of Neisseria meningitidis byinhibition of a solid phase radio-immunoassay. Infect.Immun.18:424-433.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 30                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..34                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAACGGCGGCGCCTCTGGCCAAGTTAAAGTTACTAGTAC39                                     AsnGlyGlyAlaSerGlyGlnValLysValThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AsnGlyGlyAlaSerGlyGlnValLysValThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..34                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TAGTAACTTTAACTTGGCCAGAGGCGCCGCCGTTCGTAC39                                     SerAsnPheAsnLeuAlaArgGlyAlaAlaVal                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerAsnPheAsnLeuAlaArgGlyAlaAlaVal                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GGTACGAACGGC12                                                                (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCCGTTCGTACC12                                                                (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GTTACTAGTACC12                                                                (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGTACTAGTAAC12                                                                (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..34                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GTATACCAAAGACACCAACAACAACTTGACCTTGAGTAC39                                     TyrThrLysAspThrAsnAsnAsnLeuThrLeu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TyrThrLysAspThrAsnAsnAsnLeuThrLeu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..34                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TCAAGTTCAAGTTGTTGTTGTTGTCTTTGGTATACGTAC39                                     GlnValGlnValValValValValPheGlyIle                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlnValGlnValValValValValPheGlyIle                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGTACGTATACC12                                                                (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGTATACGTACC12                                                                (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ACCTTGAGTACC12                                                                (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGTACTCAAGGT12                                                                (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 6..38                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CTAGCTATACCAAAGACACCAACAACAACTTGACCTTGT39                                     TyrThrLysAspThrAsnAsnAsnLeuThrLeu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TyrThrLysAspThrAsnAsnAsnLeuThrLeu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 6..38                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CTAGACAAGGTCAAGTTGTTGTTGGTGTCTTTGGTATAG39                                     GlnGlyGlnValValValGlyValPheGlyIle                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GlnGlyGlnValValValGlyValPheGlyIle                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ACTAGCTATACC12                                                                (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GGTATAGCTAGT12                                                                (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ACCTTGTCTAGT12                                                                (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      ACTAGACAAGGT12                                                                (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..16                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GGGCTGCAGCCTAAGTAC18                                                          GlyCysSerLeuSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyCysSerLeuSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 5..13                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TTAGGCTGCAGCCCGTAC18                                                          AlaAlaAla                                                                     (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      AlaAlaAla                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GGGCTGCAGCCTAAGTAC18                                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      TTAGGCTGCAGCCCGTAC18                                                          __________________________________________________________________________

We claim:
 1. A B cell activating molecule capable of providing immunityagainst meningococcal disease, said molecule being derived from ameningococcal, lipopolysaccharide (LPS) with at least one B cellactivating epitope, said molecule comprising the lipid A part of the LPSand at least one communal part of the oligosaccharide part (core region)of lipopolysaccharides specific for at least two meningococcalimmunotypes, wherein at least the terminal galactose residue of thelacto-N-neotetraose unit of the LPS is absent in the molecule. 2.Molecule according to claim 1, said molecule being derived from the L3core and having the following structure: ##STR10##
 3. Molecule accordingto claim 1, said molecule being derived from the L2 core and having thefollowing structure: ##STR11##
 4. A molecule according to claim 1, saidmolecule being derived from the L3 core and having the followingstructure ##STR12##
 5. A molecule according to claim 1, said moleculebeing derived from the L2 core and having the following structure##STR13##
 6. A molecule according to claim 1, wherein the meningococcalimmunotypes are L2 and L3.
 7. A B cell activating molecule capable ofproviding immunity against meningococcal disease, said molecule beingderived from a meningococcal, lipopolysaccharide (LPS) with at least oneB cell activating epitope, said molecule comprising the lipid A part ofthe LPS and at least one communal part of the oligosaccharide part (coreregion) of lipopolysaccharides specific for at least two meningococcalimmunotypes, wherein at least one galactose residue of thelacto-N-neotetraose unit of the LPS is absent in the molecule, andwherein at least one glucose residue of said lacto-N-neotetraose unit ofthe LPS is retained in the molecule.
 8. A method for preparing amolecule according to claim 1, wherein recombinant DNA techniques areused.
 9. A method according to claim 8, wherein the molecule is obtainedfrom a mutagenised production strain producing at least LPS withoutterminal galactose.
 10. A method according to claim 8, wherein themolecule is obtained from a mutagenised production strain producing atleast LPS without galactose.
 11. A method according to claim 8, whereinthe molecule is obtained from a mutagenised meningococcal productionstrain without galactose.
 12. A method according to claim 8, wherein themolecule is obtained from a mutagenised production strain without galE.13. A method according to claim 8, wherein the molecule is obtained froma mutagenised production strain without functional galE.